PROJECT SUMMARY Hearing function depends on precise connectivity patterns of spiral ganglion neurons (SGNs), olivocochlear efferents, and hair cells in the cochlea. Deficits in these connections underlie hearing impairment and the efficacy of cochlear implants. The long-term goal of this research is to define the mechanisms responsible for wiring events between neurons and hair cells in the cochlea, so that improved therapies can be developed to treat hearing loss in humans. Pou3f4 is a transcription factor expressed by otic mesenchyme cells in the cochlea and mutations in Pou3f4 cause human hearing loss. Loss of Pou3f4 in mouse models leads to morphological defects in otic mesenchyme and these models are known to be hearing impaired as a result of reduced endocochlear potential. We showed recently that Pou3f4 is critical in SGN axon guidance. But, we have a very limited understanding of how Pou3f4 controls auditory innervation mechanisms because the transcriptional targets of Pou3f4 in the cochlea are not well understood. This proposal seeks to determine the function of Pou3f4 in axon guidance, transcriptional regulation, and neuronal survival in the auditory system. Ephrins are cell-surface bound ligands that activate Ephs (receptor tyrosine kinases) to facilitate diverse forms of intercellular communication including axon guidance and synaptogenesis. Our preliminary data suggest that Pou3f4 regulates the expression of Ephrin genes in otic mesenchyme and that these are critical in hair cell wiring. Results from the proposed work will demonstrate how these Ephrin proteins control cochlear innervation and contribute innervation defects observed in Pou3f4 mutants. Results from our proposed work will also generate a comprehensive set of Pou3f4 transcriptional targets, which will include all possible factors involved in cochlear innervation. We have also found that otic mesenchyme cells express Pou3f4 into adulthood and that Pou3f4 null adults show a significant loss of SGNs. Thus, in these studies, we will also determine the mechanism(s) by which Pou3f4 normally mediates SGN survival. In this work, we will use a range of in vivo and in vitro techniques, innovative imaging approaches, and transcriptional profiling methods. The contribution of this research will be significant because it will determine how Pou3f4 and its targets contribute to the development and maintenance of the complex afferent innervation patterns within the mammalian auditory system. In addition, this work is expected to determine new guidance mechanisms required for appropriate auditory connectivity, thus it will complement ongoing work by others on neurotrophins, gene therapy, or cell replacement strategies.